Epstein Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a number of cancers including lymphomas in transplant recipients and in patients with other immunocompromising diseases. Rheumatoid arthritis and polymyositis are two autoimmune diseases in which the joints and muscles, respectively, are damaged by the body?s immune system. Patients with rheumatoid arthritis or polymyositis have a higher frequency of lymphomas that contain Epstein-Barr virus (EBV) DNA than the general population. Some of these EBV lymphomas develop during treatment of these patients with methotrexate (a drug that suppresses the immune response); several cases have been reported in which the lymphomas completely resolved when methotrexate was discontinued. Thus, methotrexate is thought to have a role in the development of some EBV-positive lymphomas. We found that methotrexate, in contrast to other immunosuppressive medications for rheumatoid arthritis or polymyositis, induced the release of infectious EBV from latently infected cells in culture. We determined that methotrexate activated two viral genes that induced the expression of other viral genes to produce virus in cell culture. Patients with rheumatoid arthritis or polymyositis who were treated with methotrexate-containing regimens had significantly higher levels of EBV DNA in their blood than patients with these diseases who were treated with immunosuppressive regimens that did not include methotrexate.